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Patrick J Venables - One of the best experts on this subject based on the ideXlab platform.
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autoimmunity to specific citrullinated proteins gives the first clues to the etiology of rheumatoid arthritis
Immunological Reviews, 2010Co-Authors: Natalia Wegner, Benjamin A Fisher, Vivianne Malmstrom, Karin Lundberg, Andrew J Kinloch, Marc Feldmann, Patrick J VenablesAbstract:: Rheumatoid arthritis (RA) is now clearly a true autoimmune disease with accumulating evidence of pathogenic disease-specific autoimmunity to citrullinated proteins. Citrullination, also termed deimination, is a modification of arginine side chains catalyzed by peptidylarginine deiminase (PAD) enzymes. This post-translational modification has the potential to alter the structure, antigenicity, and function of proteins. In RA, antibodies to cyclic citrullinated peptides are now well established for clinical diagnosis, though we argue that the identification of specific citrullinated antigens, as whole proteins, is necessary for exploring pathogenic mechanisms. Four citrullinated antigens, fibrinogen, vimentin, collagen type II, and Alpha-Enolase, are now well established, with others awaiting further characterization. All four proteins are expressed in the joint, and there is evidence that antibodies to citrullinated fibrinogen and collagen type II mediate inflammation by the formation of immune complexes, both in humans and animal models. Antibodies to citrullinated proteins are associated with HLA 'shared epitope' alleles, and autoimmunity to at least one antigenic sequence, the CEP-1 peptide from citrullinated Alpha-Enolase (KIHAcitEIFDScitGNPTVE), shows a specific association with HLA-DRB1*0401, *0404, 620W PTPN22, and smoking. Periodontitis, in which Porphyromonas gingivalis is a major pathogenic bacterium, has been linked to RA in epidemiological studies and also shares similar gene/environment associations. This is also the only bacterium identified that expresses endogenous citrullinated proteins and its own bacterial PAD enzyme, though the precise molecular mechanisms of bacterial citrullination have yet to be explored. Thus, both smoking and Porphyromonas gingivalis are attractive etiological agents for further investigation into the gene/environment/autoimmunity triad of RA.
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antibodies to citrullinated alpha enolase peptide 1 are specific for rheumatoid arthritis and cross react with bacterial enolase
Arthritis & Rheumatism, 2008Co-Authors: Karin Lundberg, Benjamin A Fisher, P Charles, Natalia Wegner, Andrew J Kinloch, Robin Wait, Ted R Mikuls, Patrick J VenablesAbstract:Objective To map the antibody response to human citrullinated α-enolase, a candidate autoantigen in rheumatoid arthritis (RA), and to examine cross-reactivity with bacterial enolase. Methods Serum samples obtained from patients with RA, disease control subjects, and healthy control subjects were tested by enzyme-linked immunosorbent assay (ELISA) for reactivity with citrullinated α-enolase peptides. Antibodies specific for the immunodominant epitope were raised in rabbits or were purified from RA sera. Cross-reactivity with other citrullinated epitopes was investigated by inhibition ELISAs, and cross-reactivity with bacterial enolase was investigated by immunoblotting. Results An immunodominant peptide, citrullinated α-enolase peptide 1, was identified. Antibodies to this epitope were observed in 37–62% of sera obtained from patients with RA, 3% of sera obtained from disease control subjects, and 2% of sera obtained from healthy control subjects. Binding was inhibited with homologous peptide but not with the arginine-containing control peptide or with 4 citrullinated peptides from elsewhere on the molecule, indicating that antibody binding was dependent on both citrulline and flanking amino acids. The immunodominant peptide showed 82% homology with enolase from Porphyromonas gingivalis, and the levels of antibodies to citrullinated α-enolase peptide 1 correlated with the levels of antibodies to the bacterial peptide (r2 = 0.803, P < 0.0001). Affinity-purified antibodies to the human peptide cross-reacted with citrullinated recombinant P gingivalis enolase. Conclusion We have identified an immunodominant epitope in citrullinated α-enolase, to which antibodies are specific for RA. Our data on sequence similarity and cross-reactivity with bacterial enolase may indicate a role for bacterial infection, particularly with P gingivalis, in priming autoimmunity in a subset of patients with RA.
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synovial fluid is a site of citrullination of autoantigens in inflammatory arthritis
Arthritis & Rheumatism, 2008Co-Authors: Andrew J Kinloch, Vivianne Malmstrom, Karin Lundberg, Natalia Wegner, Robin Wait, A J W Zendman, Tore Saxne, Patrick J VenablesAbstract:Objective. To examine synovial fluid as a site for generating citrullinated antigens, including the candidate autoantigen citrullinated Alpha-Enolase, in rheumatoid arthritis (RA). Methods. Synovial fluid was obtained from 20 patients with RA, 20 patients with spondylarthritides (SpA), and 20 patients with osteoarthritis (OA). Samples were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by staining with Coomassie blue and immunoblotting for citrullinated proteins, Alpha-Enolase, and the deiminating enzymes peptidylarginine deiminase type 2 (PAD-2) and PAD-4. Proteins from an RA synovial fluid sample were separated by 2-dimensional electrophoresis, and each protein was identified by immunoblotting and mass spectrometry. Antibodies to citrullinated Alpha-Enolase peptide 1 (CEP-1) and cyclic citrullinated peptide 2 were measured by enzyme-linked immunosorbent assay. Results. Citrullinated polypeptides were detected in the synovial fluid from patients with RA and patients with SpA, but not in OA samples. Alpha-Enolase was detected in all of the samples, with mean levels of 6.4 ng/mu l in RA samples, 4.3 ng/mu l in SpA samples, and <0.9 ng/mu l in OA samples. Two-dimensional electrophoresis provided evidence that the Alpha-Enolase was citrullinated in RA synovial fluid. The citrullinating enzyme PAD-4 was detected in samples from all 3 disease groups. PAD-2 was detected in 18 of the RA samples, in 1.6 of the SpA samples, and in none of the OA samples. Antibodies to CEP-1 were found in 12 of the RA samples (60%), in none of the SpA samples, and in I OA sample. Conclusion. These results highlight the importance of synovial fluid for the expression of citrullinated autoantigens in inflammatory arthritis. Whereas the expression of citrullinated proteins is a product of inflammation, the antibody response remains specific for RA.
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identification of citrullinated alpha enolase as a candidate autoantigen in rheumatoid arthritis
Arthritis Research & Therapy, 2005Co-Authors: Andrew J Kinloch, Karin Lundberg, Robin Wait, Verena Tatzer, David Peston, Phillipe Donatien, David L Moyes, Peter C Taylor, Patrick J VenablesAbstract:Antibodies against citrullinated proteins are highly specific for rheumatoid arthritis (RA), but little is understood about their citrullinated target antigens. We have detected a candidate citrullinated protein by immunoblotting lysates of monocytic and granulocytic HL-60 cells treated with peptidylarginine deiminase. In an initial screen of serum samples from four patients with RA and one control, a protein of molecular mass 47 kDa from monocytic HL-60s reacted with sera from the patients, but not with the serum from the control. Only the citrullinated form of the protein was recognised. The antigen was identified by tandem mass spectrometry as α-enolase, and the positions of nine citrulline residues in the sequence were determined. Serum samples from 52 patients with RA and 40 healthy controls were tested for presence of antibodies against citrullinated and non-citrullinated α-enolase by immunoblotting of the purified antigens. Twenty-four sera from patients with RA (46%) reacted with citrullinated α-enolase, of which seven (13%) also recognised the non-citrullinated protein. Six samples from the controls (15%) reacted with both forms. α-Enolase was detected in the RA joint, where it co-localised with citrullinated proteins. The presence of antibody together with expression of antigen within the joint implicates citrullinated α-enolase as a candidate autoantigen that could drive the chronic inflammatory response in RA.
Ja Gerlt - One of the best experts on this subject based on the ideXlab platform.
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evolution of enzymatic activities in the enolase superfamily l rhamnonate dehydratase
Biochemistry, 2008Co-Authors: John F. Rakus, Alexander A Fedorov, E V Fedorov, Margaret E Glasner, Brian K Hubbard, Joseph D Delli, Patricia C Babbitt, Steven C Almo, Ja GerltAbstract:The l-rhamnonate dehydratase (RhamD) function was assigned to a previously uncharacterized family in the mechanistically diverse enolase superfamily that is encoded by the genome of Escherichia coli K-12. We screened a library of acid sugars to discover that the enzyme displays a promiscuous substrate specificity: l-rhamnonate (6-deoxy- l-mannonate) has the "best" kinetic constants, with l-mannonate, l-lyxonate, and d-gulonate dehydrated less efficiently. Crystal structures of the RhamDs from both E. coli K-12 and Salmonella typhimurium LT2 (95% sequence identity) were obtained in the presence of Mg (2+); the structure of the RhamD from S. typhimurium was also obtained in the presence of 3-deoxy- l-rhamnonate (obtained by reduction of the product with NaBH 4). Like other members of the enolase superfamily, RhamD contains an N-terminal alpha + beta capping domain and a C-terminal (beta/alpha) 7beta-barrel (modified TIM-barrel) catalytic domain with the active site located at the interface between the two domains. In contrast to other members, the specificity-determining "20s loop" in the capping domain is extended in length and the "50s loop" is truncated. The ligands for the Mg (2+) are Asp 226, Glu 252 and Glu 280 located at the ends of the third, fourth and fifth beta-strands, respectively. The active site of RhamD contains a His 329-Asp 302 dyad at the ends of the seventh and sixth beta-strands, respectively, with His 329 positioned to function as the general base responsible for abstraction of the C2 proton of l-rhamnonate to form a Mg (2+)-stabilized enediolate intermediate. However, the active site does not contain other acid/base catalysts that have been implicated in the reactions catalyzed by other members of the MR subgroup of the enolase superfamily. Based on the structure of the liganded complex, His 329 also is expected to function as the general acid that both facilitates departure of the 3-OH group in a syn-dehydration reaction and delivers a proton to carbon-3 to replace the 3-OH group with retention of configuration.
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evolution of enzymatic activities in the enolase superfamily l talarate galactarate dehydratase from salmonella typhimurium lt2
Biochemistry, 2007Co-Authors: Wen Shan Yew, Alexander A Fedorov, E V Fedorov, Ja GerltAbstract:We assigned l-talarate dehydratase (TalrD) and galactarate dehydratase (GalrD) functions to a group of orthologous proteins in the mechanistically diverse enolase superfamily, focusing our characterization on the protein encoded by the Salmonella typhimurium LT2 genome (GI:16766982; STM3697). Like the homologous mandelate racemase, l-fuconate dehydratase, and d-tartrate dehydratase, the active site of TalrD/GalrD contains a general acid/base Lys 197 at the end of the second beta-strand in the (beta/alpha)7beta-barrel domain, Asp 226, Glu 252, and Glu 278 as ligands for the essential Mg2+ at the ends of the third, fourth, and fifth beta-strands, a general acid/base His 328-Asp 301 dyad at the ends of the seventh and sixth beta-strands, and an electrophilic Glu 348 at the end of the eighth beta-strand. We discovered the function of STM3697 by screening a library of acid sugars; it catalyzes the efficient dehydration of both l-talarate (kcat = 2.1 s-1, kcat/Km = 9.1 x 10(3) M-1 s-1) and galactarate (kcat = 3.5 s-1, kcat/Km = 1.1 x 10(4) M-1 s-1). Because l-talarate is a previously unknown metabolite, we demonstrated that S. typhimurium LT2 can utilize l-talarate as carbon source. Insertional disruption of the gene encoding STM3697 abolishes this phenotype; this disruption also diminishes, but does not eliminate, the ability of the organism to utilize galactarate as carbon source. The dehydration of l-talarate is accompanied by competing epimerization to galactarate; little epimerization to l-talarate is observed in the dehydration of galactarate. On the basis of (1) structures of the wild type enzyme complexed with l-lyxarohydroxamate, an analogue of the enolate intermediate, and of the K197A mutant complexed with l-glucarate, a substrate for exchange of the alpha-proton, and (2) incorporation of solvent deuterium into galactarate in competition with dehydration, we conclude that Lys 197 functions as the galactarate-specific base and His 328 functions as the l-talarate-specific base. The epimerization of l-talarate to galactarate that competes with dehydration can be rationalized by partitioning of the enolate intermediate between dehydration (departure of the 3-OH group catalyzed by the conjugate acid of His 328) and epimerization (protonation on C2 by the conjugate acid of Lys 197). The promiscuous catalytic activities discovered for STM3697 highlight the evolutionary potential of a "conserved" active site architecture.
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evolution of enzymatic activities in the enolase superfamily n succinylamino acid racemase and a new pathway for the irreversible conversion of d to l amino acids
Biochemistry, 2006Co-Authors: Ayano Sakai, Dao Feng Xiang, Ling Song, Wen Shan Yew, Frank M. Raushel, Ja GerltAbstract:Members of the mechanistically diverse enolase superfamily catalyze reactions that are initiated by abstraction of the alpha-proton of a carboxylate anion to generate an enolate anion intermediate that is stabilized by coordination to a Mg2+ ion. The catalytic groups, ligands for an essential Mg2+ and acid/base catalysts, are located in the (beta/alpha)8-barrel domain of the bidomain proteins. The assigned physiological functions in the muconate lactonizing enzyme (MLE) subgroup (Lys acid/base catalysts at the ends of the second and sixth beta-strands in the barrel domain) are cycloisomerization (MLE), dehydration (o-succinylbenzoate synthase; OSBS), and epimerization (L-Ala-D/L-Glu epimerase). We previously studied a putatively promiscuous member of the MLE subgroup with uncertain physiological function from Amycolatopsis that was discovered based on its ability to catalyze the racemization of N-acylamino acids (N-acylamino acid racemase; NAAAR) but also catalyzes the OSBS reaction [OSBS/NAAAR; Palmer, D. R., Garrett, J. B., Sharma, V., Meganathan, R., Babbitt, P. C., and Gerlt, J. A. (1999) Biochemistry 38, 4252-4258]. In this manuscript, we report functional characterization of a homologue of this protein encoded by the genome of Geobacillus kaustophilus as well as two other proteins that are encoded by the same operon, a divergent member of the Gcn5-related N-acetyltransferase (GNAT) superfamily of enzymes whose members catalyze the transfer an acyl group from an acyl-CoA donor to an amine acceptor, and a member of the M20 peptidase/carboxypeptidase G2 family. We determined that the member of the GNAT superfamily is succinyl-CoA:D-amino acid N-succinyltransferase, the member of the enolase superfamily is N-succinylamino acid racemase (NSAR), and the member of the M20 peptidase/carboxypeptidase G2 family is N-succinyl-L-amino acid hydrolase. We conclude that (1) these enzymes constitute a novel, irreversible pathway for the conversion of D- to L-amino acids and (2) the NSAR reaction is a new physiological function in the MLE subgroup. The NSAR is also functionally promiscuous and catalyzes an efficient OSBS reaction; intriguingly, the operon for menaquinone biosynthesis in G. kaustophilus does not encode an OSBS, raising the possibility that the NSAR is a bifunctional enzyme rather than an accidentally promiscuous enzyme.
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evolution of enzymatic activity in the enolase superfamily structural studies of the promiscuous o succinylbenzoate synthase from amycolatopsis
Biochemistry, 2004Co-Authors: Erika Taylor A Ringia, James B. Garrett, James B Thoden, Hazel M Holden, Ivan Rayment, Ja GerltAbstract:o-Succinylbenzoate synthase (OSBS) from Amycolatopsis, a member of the enolase superfamily, catalyzes the Mn2+-dependent exergonic dehydration of 2-succinyl-6R-hydroxy-2,4-cyclohexadiene-1R-carboxylate (SHCHC) to 4-(2'-carboxylphenyl)-4-oxobutyrate (o-succinylbenzoate or OSB) in the menaquinone biosynthetic pathway. This enzyme first was identified as an N-acylamino acid racemase (NAAAR), with the optimal substrates being the enantiomers of N-acetyl methionine. This laboratory subsequently discovered that this protein is a much better catalyst of the OSBS reaction, with the value of k(cat)/K(M), for dehydration, 2.5 x 10(5) M(-1) s(-1), greatly exceeding that for 1,1-proton transfer using the enantiomers of N-acetylmethionine as substrate, 3.1 x 10(2) M(-1) s(-1) [Palmer, D. R., Garrett, J. B., Sharma, V., Meganathan, R., Babbitt, P. C., and Gerlt, J. A. (1999) Biochemistry 38, 4252-8]. The efficiency of the promiscuous NAAAR reaction is enhanced with alternate substrates whose structures mimic that of the SHCHC substrate for the OSBS reaction, for example, the value of k(cat)/K(M) for the enantiomers of N-succinyl phenylglycine, 2.0 x 10(5) M(-1) s(-1), is comparable to that for the OSBS reaction. The mechanisms of the NAAAR and OSBS reactions have been explored using mutants of Lys 163 and Lys 263 (K163A/R/S and K263A/R/S), the putative acid/base catalysts identified by sequence alignments with other OSBSs, including the structurally characterized OSBS from Escherichia coli. Although none of the mutants display detectable OSBS or NAAAR activities, K163R and K163S catalyze stereospecific exchange of the alpha-hydrogen of N-succinyl-(S)-phenylglycine with solvent hydrogen, and K263R and K263 catalyze the stereospecific exchange the alpha-hydrogen of N-succinyl-(R)-phenylglycine, consistent with formation of a Mn2+-stabilized enolate anion intermediate. The rates of the exchange reactions catalyzed by the wild-type enzyme exceed those for racemization. That this enzyme can catalyze two different reactions, each involving a stabilized enediolate anion intermediate, supports the hypothesis that evolution of function in the enolase superfamily proceeds by pathways involving functional promiscuity.
Moitza Principe - One of the best experts on this subject based on the ideXlab platform.
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alpha enolase eno1 controls alpha v beta 3 integrin expression and regulates pancreatic cancer adhesion invasion and metastasis
Journal of Hematology & Oncology, 2017Co-Authors: Moitza Principe, Michela Capello, Michelle Samuel Chattaragada, Simone Borgoni, Mariafrancesca Cascione, Sara Bulfamante, Jennifer Chapelle, Francesca Di Modugno, Sammy Ferriborgogno, Paola DefilippiAbstract:We have previously shown that in pancreatic ductal adenocarcinoma (PDA) cells, the glycolytic enzyme Alpha-Enolase (ENO1) also acts as a plasminogen receptor and promotes invasion and metastasis formation. Moreover, ENO1 silencing in PDA cells induces oxidative stress, senescence and profoundly modifies PDA cell metabolism. Although anti-ENO1 antibody inhibits PDA cell migration and invasion, little is known about the role of ENO1 in regulating cell-cell and cell-matrix contacts. We therefore investigated the effect of ENO1 silencing on the modulation of cell morphology, adhesion to matrix substrates, cell invasiveness, and metastatic ability. The membrane and cytoskeleton modifications that occurred in ENO1-silenced (shENO1) PDA cells were investigated by a combination of confocal microscopy and atomic force microscopy (AFM). The effect of ENO1 silencing was then evaluated by phenotypic and functional experiments to identify the role of ENO1 in adhesion, migration, and invasion, as well as in senescence and apoptosis. The experimental results were then validated in a mouse model. We observed a significant increase in the roughness of the cell membrane due to ENO1 silencing, a feature associated with an impaired ability to migrate and invade, along with a significant downregulation of proteins involved in cell-cell and cell-matrix adhesion, including alpha v/beta 3 integrin in shENO1 PDA cells. These changes impaired the ability of shENO1 cells to adhere to Collagen I and IV and Fibronectin and caused an increase in RGD-independent adhesion to vitronectin (VN) via urokinase plasminogen activator receptor (uPAR). Binding of uPAR to VN triggers integrin-mediated signals, which result in ERK1-2 and RAC activation, accumulation of ROS, and senescence. In shENO1 cancer cells, the use of an anti-uPAR antibody caused significant reduction of ROS production and senescence. Overall, a decrease of in vitro and in vivo cell migration and invasion of shENO1 PDA cells was observed. These data demonstrate that ENO1 promotes PDA survival, migration, and metastasis through cooperation with integrins and uPAR.
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Alpha-Enolase (ENO1) controls alpha v/beta 3 integrin expression and regulates pancreatic cancer adhesion, invasion, and metastasis
Journal of Hematology & Oncology, 2017Co-Authors: Moitza Principe, Michela Capello, Michelle Samuel Chattaragada, Simone Borgoni, Mariafrancesca Cascione, Sammy Ferri-borgogno, Sara Bulfamante, Jennifer Chapelle, Francesca Di Modugno, Paola DefilippiAbstract:Background We have previously shown that in pancreatic ductal adenocarcinoma (PDA) cells, the glycolytic enzyme Alpha-Enolase (ENO1) also acts as a plasminogen receptor and promotes invasion and metastasis formation. Moreover, ENO1 silencing in PDA cells induces oxidative stress, senescence and profoundly modifies PDA cell metabolism. Although anti-ENO1 antibody inhibits PDA cell migration and invasion, little is known about the role of ENO1 in regulating cell-cell and cell-matrix contacts. We therefore investigated the effect of ENO1 silencing on the modulation of cell morphology, adhesion to matrix substrates, cell invasiveness, and metastatic ability. Methods The membrane and cytoskeleton modifications that occurred in ENO1-silenced (shENO1) PDA cells were investigated by a combination of confocal microscopy and atomic force microscopy (AFM). The effect of ENO1 silencing was then evaluated by phenotypic and functional experiments to identify the role of ENO1 in adhesion, migration, and invasion, as well as in senescence and apoptosis. The experimental results were then validated in a mouse model. Results We observed a significant increase in the roughness of the cell membrane due to ENO1 silencing, a feature associated with an impaired ability to migrate and invade, along with a significant downregulation of proteins involved in cell-cell and cell-matrix adhesion, including alpha v/beta 3 integrin in shENO1 PDA cells. These changes impaired the ability of shENO1 cells to adhere to Collagen I and IV and Fibronectin and caused an increase in RGD-independent adhesion to vitronectin (VN) via urokinase plasminogen activator receptor (uPAR). Binding of uPAR to VN triggers integrin-mediated signals, which result in ERK1-2 and RAC activation, accumulation of ROS, and senescence. In shENO1 cancer cells, the use of an anti-uPAR antibody caused significant reduction of ROS production and senescence. Overall, a decrease of in vitro and in vivo cell migration and invasion of shENO1 PDA cells was observed. Conclusion These data demonstrate that ENO1 promotes PDA survival, migration, and metastasis through cooperation with integrins and uPAR.
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abstract a65 effects of alpha enolase silencing in adenocarcinoma pancreatic cell line
Cancer Research, 2012Co-Authors: Moitza Principe, Michela Capello, Michelle Samuel Chattaragada, Weidong Zhou, Lance A Liotta, Emanuel F Petricoin, Francesco NovelliAbstract:Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy characterized by rapid progression, invasiveness, and resistance to treatment. Alpha-Enolase (ENOA) is a key glycolytic enzyme that is also expressed on the surface of immune and tumor cells where it acts as a plasminogen receptor. In our laboratory it has been demonstrated that ENOA is expressed on the surface of several PDAC cell lines where is crucial for plasminogen-dependent invasion. A monoclonal antibody against ENOA inhibits this effect both in vitro and in vivo (unpublished data). Aim: We investigated the effect of ENOA silencing in PDAC cell line in order to identify key proteins that are different modulated and clarify its multifunctional role in pancreatic tumorigenesis. Methods: CF-PAC-1 human PDAC cell line was infected with a lentiviral vector delivering a short hairpin RNA (shRNA) targeting ENOA 3’UTR or a scrambled shRNA as a control. After silencing, ENOA mRNA was evaluated by real-time PCR and protein level was assessed by and Western Blot Analysis in each subcellular fraction (cytosolic, membrane and nuclear protein). Proliferation was monitored by cell counting and MTT assays. The cell cycle analysis was performed by cytofluorimetry. A pyruvate assay kit was used to determinate pyruvate production. The tumorigenicity of ENOA silenced cells was assessed by a colony forming assay and their invasive capability was tested by a transwell matrigel invasion assay. Adhesion capability of the cells was evaluated by plate fixing and cell counting. Finally the proteomes of ENOA silenced and control (scrambled or normal CF-PAC-1) cells were compared by LTQ-Orbitrap tandem mass spectrometry analysis and spectra count label-free quantification approach. Results: After lentiviral infection, ENOA was silenced both at the transcript and at the protein level by around 80%, and the silencing was evident in all the cellular fractions. Compared to the control cells, ENOA silenced cells displayed a delay in proliferation and a decreased survival capability, even if the pyruvate production was not affected. This effect was due to a partially block of cell cycle in G2/M phase and not to an apoptotic events. In silenced cells, the capability to form colonies in soft agar was reduced to 40-50%, the invasion in response to plasminogen was abolished, and the adhesive capability decreased to 40%. Finally, more than 1500 proteins were identified from each sample by tandem mass spectrometry analysis and a large number of proteins were differentially expressed in ENOA silenced cells compared to control cells. These proteins were mainly involved in cell adhesion, cell metabolism and cell proliferation and mRNA analysis revealed that the most significantly downregulated proteins (cell-cell and cell-matrix adhesion regulators) were controlled either at the transcriptional or post-translational levels. Conclusions: Together, this data confirm that ENOA in PDAC is not only a glycolytic enzyme, but is a key regulator of a broad spectrum of pathways involved in tumorigenesis. The mechanisms through which ENOA controls cell adhesion, proliferation and metabolism in PDAC are currently under investigation. Citation Format: Moitza Principe, Michela Capello, Michelle Samuel Chattaragada, Weidong Zhou, Lance Liotta, Emanuel Petricoin, Francesco Novelli. Effects of Alpha-Enolase silencing in adenocarcinoma pancreatic cell line. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A65.
Paola Defilippi - One of the best experts on this subject based on the ideXlab platform.
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Alpha-Enolase (ENO1) controls alpha v/beta 3 integrin expression and regulates pancreatic cancer adhesion, invasion, and metastasis
Journal of Hematology & Oncology, 2017Co-Authors: Moitza Principe, Michela Capello, Michelle Samuel Chattaragada, Simone Borgoni, Mariafrancesca Cascione, Sammy Ferri-borgogno, Sara Bulfamante, Jennifer Chapelle, Francesca Di Modugno, Paola DefilippiAbstract:Background We have previously shown that in pancreatic ductal adenocarcinoma (PDA) cells, the glycolytic enzyme Alpha-Enolase (ENO1) also acts as a plasminogen receptor and promotes invasion and metastasis formation. Moreover, ENO1 silencing in PDA cells induces oxidative stress, senescence and profoundly modifies PDA cell metabolism. Although anti-ENO1 antibody inhibits PDA cell migration and invasion, little is known about the role of ENO1 in regulating cell-cell and cell-matrix contacts. We therefore investigated the effect of ENO1 silencing on the modulation of cell morphology, adhesion to matrix substrates, cell invasiveness, and metastatic ability. Methods The membrane and cytoskeleton modifications that occurred in ENO1-silenced (shENO1) PDA cells were investigated by a combination of confocal microscopy and atomic force microscopy (AFM). The effect of ENO1 silencing was then evaluated by phenotypic and functional experiments to identify the role of ENO1 in adhesion, migration, and invasion, as well as in senescence and apoptosis. The experimental results were then validated in a mouse model. Results We observed a significant increase in the roughness of the cell membrane due to ENO1 silencing, a feature associated with an impaired ability to migrate and invade, along with a significant downregulation of proteins involved in cell-cell and cell-matrix adhesion, including alpha v/beta 3 integrin in shENO1 PDA cells. These changes impaired the ability of shENO1 cells to adhere to Collagen I and IV and Fibronectin and caused an increase in RGD-independent adhesion to vitronectin (VN) via urokinase plasminogen activator receptor (uPAR). Binding of uPAR to VN triggers integrin-mediated signals, which result in ERK1-2 and RAC activation, accumulation of ROS, and senescence. In shENO1 cancer cells, the use of an anti-uPAR antibody caused significant reduction of ROS production and senescence. Overall, a decrease of in vitro and in vivo cell migration and invasion of shENO1 PDA cells was observed. Conclusion These data demonstrate that ENO1 promotes PDA survival, migration, and metastasis through cooperation with integrins and uPAR.
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alpha enolase eno1 controls alpha v beta 3 integrin expression and regulates pancreatic cancer adhesion invasion and metastasis
Journal of Hematology & Oncology, 2017Co-Authors: Moitza Principe, Michela Capello, Michelle Samuel Chattaragada, Simone Borgoni, Mariafrancesca Cascione, Sara Bulfamante, Jennifer Chapelle, Francesca Di Modugno, Sammy Ferriborgogno, Paola DefilippiAbstract:We have previously shown that in pancreatic ductal adenocarcinoma (PDA) cells, the glycolytic enzyme Alpha-Enolase (ENO1) also acts as a plasminogen receptor and promotes invasion and metastasis formation. Moreover, ENO1 silencing in PDA cells induces oxidative stress, senescence and profoundly modifies PDA cell metabolism. Although anti-ENO1 antibody inhibits PDA cell migration and invasion, little is known about the role of ENO1 in regulating cell-cell and cell-matrix contacts. We therefore investigated the effect of ENO1 silencing on the modulation of cell morphology, adhesion to matrix substrates, cell invasiveness, and metastatic ability. The membrane and cytoskeleton modifications that occurred in ENO1-silenced (shENO1) PDA cells were investigated by a combination of confocal microscopy and atomic force microscopy (AFM). The effect of ENO1 silencing was then evaluated by phenotypic and functional experiments to identify the role of ENO1 in adhesion, migration, and invasion, as well as in senescence and apoptosis. The experimental results were then validated in a mouse model. We observed a significant increase in the roughness of the cell membrane due to ENO1 silencing, a feature associated with an impaired ability to migrate and invade, along with a significant downregulation of proteins involved in cell-cell and cell-matrix adhesion, including alpha v/beta 3 integrin in shENO1 PDA cells. These changes impaired the ability of shENO1 cells to adhere to Collagen I and IV and Fibronectin and caused an increase in RGD-independent adhesion to vitronectin (VN) via urokinase plasminogen activator receptor (uPAR). Binding of uPAR to VN triggers integrin-mediated signals, which result in ERK1-2 and RAC activation, accumulation of ROS, and senescence. In shENO1 cancer cells, the use of an anti-uPAR antibody caused significant reduction of ROS production and senescence. Overall, a decrease of in vitro and in vivo cell migration and invasion of shENO1 PDA cells was observed. These data demonstrate that ENO1 promotes PDA survival, migration, and metastasis through cooperation with integrins and uPAR.
Andrew J Kinloch - One of the best experts on this subject based on the ideXlab platform.
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autoimmunity to specific citrullinated proteins gives the first clues to the etiology of rheumatoid arthritis
Immunological Reviews, 2010Co-Authors: Natalia Wegner, Benjamin A Fisher, Vivianne Malmstrom, Karin Lundberg, Andrew J Kinloch, Marc Feldmann, Patrick J VenablesAbstract:: Rheumatoid arthritis (RA) is now clearly a true autoimmune disease with accumulating evidence of pathogenic disease-specific autoimmunity to citrullinated proteins. Citrullination, also termed deimination, is a modification of arginine side chains catalyzed by peptidylarginine deiminase (PAD) enzymes. This post-translational modification has the potential to alter the structure, antigenicity, and function of proteins. In RA, antibodies to cyclic citrullinated peptides are now well established for clinical diagnosis, though we argue that the identification of specific citrullinated antigens, as whole proteins, is necessary for exploring pathogenic mechanisms. Four citrullinated antigens, fibrinogen, vimentin, collagen type II, and Alpha-Enolase, are now well established, with others awaiting further characterization. All four proteins are expressed in the joint, and there is evidence that antibodies to citrullinated fibrinogen and collagen type II mediate inflammation by the formation of immune complexes, both in humans and animal models. Antibodies to citrullinated proteins are associated with HLA 'shared epitope' alleles, and autoimmunity to at least one antigenic sequence, the CEP-1 peptide from citrullinated Alpha-Enolase (KIHAcitEIFDScitGNPTVE), shows a specific association with HLA-DRB1*0401, *0404, 620W PTPN22, and smoking. Periodontitis, in which Porphyromonas gingivalis is a major pathogenic bacterium, has been linked to RA in epidemiological studies and also shares similar gene/environment associations. This is also the only bacterium identified that expresses endogenous citrullinated proteins and its own bacterial PAD enzyme, though the precise molecular mechanisms of bacterial citrullination have yet to be explored. Thus, both smoking and Porphyromonas gingivalis are attractive etiological agents for further investigation into the gene/environment/autoimmunity triad of RA.
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antibodies to citrullinated alpha enolase peptide 1 are specific for rheumatoid arthritis and cross react with bacterial enolase
Arthritis & Rheumatism, 2008Co-Authors: Karin Lundberg, Benjamin A Fisher, P Charles, Natalia Wegner, Andrew J Kinloch, Robin Wait, Ted R Mikuls, Patrick J VenablesAbstract:Objective To map the antibody response to human citrullinated α-enolase, a candidate autoantigen in rheumatoid arthritis (RA), and to examine cross-reactivity with bacterial enolase. Methods Serum samples obtained from patients with RA, disease control subjects, and healthy control subjects were tested by enzyme-linked immunosorbent assay (ELISA) for reactivity with citrullinated α-enolase peptides. Antibodies specific for the immunodominant epitope were raised in rabbits or were purified from RA sera. Cross-reactivity with other citrullinated epitopes was investigated by inhibition ELISAs, and cross-reactivity with bacterial enolase was investigated by immunoblotting. Results An immunodominant peptide, citrullinated α-enolase peptide 1, was identified. Antibodies to this epitope were observed in 37–62% of sera obtained from patients with RA, 3% of sera obtained from disease control subjects, and 2% of sera obtained from healthy control subjects. Binding was inhibited with homologous peptide but not with the arginine-containing control peptide or with 4 citrullinated peptides from elsewhere on the molecule, indicating that antibody binding was dependent on both citrulline and flanking amino acids. The immunodominant peptide showed 82% homology with enolase from Porphyromonas gingivalis, and the levels of antibodies to citrullinated α-enolase peptide 1 correlated with the levels of antibodies to the bacterial peptide (r2 = 0.803, P < 0.0001). Affinity-purified antibodies to the human peptide cross-reacted with citrullinated recombinant P gingivalis enolase. Conclusion We have identified an immunodominant epitope in citrullinated α-enolase, to which antibodies are specific for RA. Our data on sequence similarity and cross-reactivity with bacterial enolase may indicate a role for bacterial infection, particularly with P gingivalis, in priming autoimmunity in a subset of patients with RA.
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synovial fluid is a site of citrullination of autoantigens in inflammatory arthritis
Arthritis & Rheumatism, 2008Co-Authors: Andrew J Kinloch, Vivianne Malmstrom, Karin Lundberg, Natalia Wegner, Robin Wait, A J W Zendman, Tore Saxne, Patrick J VenablesAbstract:Objective. To examine synovial fluid as a site for generating citrullinated antigens, including the candidate autoantigen citrullinated Alpha-Enolase, in rheumatoid arthritis (RA). Methods. Synovial fluid was obtained from 20 patients with RA, 20 patients with spondylarthritides (SpA), and 20 patients with osteoarthritis (OA). Samples were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by staining with Coomassie blue and immunoblotting for citrullinated proteins, Alpha-Enolase, and the deiminating enzymes peptidylarginine deiminase type 2 (PAD-2) and PAD-4. Proteins from an RA synovial fluid sample were separated by 2-dimensional electrophoresis, and each protein was identified by immunoblotting and mass spectrometry. Antibodies to citrullinated Alpha-Enolase peptide 1 (CEP-1) and cyclic citrullinated peptide 2 were measured by enzyme-linked immunosorbent assay. Results. Citrullinated polypeptides were detected in the synovial fluid from patients with RA and patients with SpA, but not in OA samples. Alpha-Enolase was detected in all of the samples, with mean levels of 6.4 ng/mu l in RA samples, 4.3 ng/mu l in SpA samples, and <0.9 ng/mu l in OA samples. Two-dimensional electrophoresis provided evidence that the Alpha-Enolase was citrullinated in RA synovial fluid. The citrullinating enzyme PAD-4 was detected in samples from all 3 disease groups. PAD-2 was detected in 18 of the RA samples, in 1.6 of the SpA samples, and in none of the OA samples. Antibodies to CEP-1 were found in 12 of the RA samples (60%), in none of the SpA samples, and in I OA sample. Conclusion. These results highlight the importance of synovial fluid for the expression of citrullinated autoantigens in inflammatory arthritis. Whereas the expression of citrullinated proteins is a product of inflammation, the antibody response remains specific for RA.
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identification of citrullinated alpha enolase as a candidate autoantigen in rheumatoid arthritis
Arthritis Research & Therapy, 2005Co-Authors: Andrew J Kinloch, Karin Lundberg, Robin Wait, Verena Tatzer, David Peston, Phillipe Donatien, David L Moyes, Peter C Taylor, Patrick J VenablesAbstract:Antibodies against citrullinated proteins are highly specific for rheumatoid arthritis (RA), but little is understood about their citrullinated target antigens. We have detected a candidate citrullinated protein by immunoblotting lysates of monocytic and granulocytic HL-60 cells treated with peptidylarginine deiminase. In an initial screen of serum samples from four patients with RA and one control, a protein of molecular mass 47 kDa from monocytic HL-60s reacted with sera from the patients, but not with the serum from the control. Only the citrullinated form of the protein was recognised. The antigen was identified by tandem mass spectrometry as α-enolase, and the positions of nine citrulline residues in the sequence were determined. Serum samples from 52 patients with RA and 40 healthy controls were tested for presence of antibodies against citrullinated and non-citrullinated α-enolase by immunoblotting of the purified antigens. Twenty-four sera from patients with RA (46%) reacted with citrullinated α-enolase, of which seven (13%) also recognised the non-citrullinated protein. Six samples from the controls (15%) reacted with both forms. α-Enolase was detected in the RA joint, where it co-localised with citrullinated proteins. The presence of antibody together with expression of antigen within the joint implicates citrullinated α-enolase as a candidate autoantigen that could drive the chronic inflammatory response in RA.